The invention in the field of biochemistry and cell and molecular biology relates to novel receptor-type protein tyrosine phosphatase proteins or glycoproteins, termed RPTPxcex1, RPTPxcex2 and RPTPxcex3 (also designated R-PTPase-xcex1, xcex2 and xcex3), DNA coding therefor, methods for production and identification of the proteins, and methods for screening compounds capable of binding to and inhibiting or stimulating PTPase enzymatic activity.
The identification of several growth factor receptors and retroviral oncogenes as tyrosine-specific protein kinases indicated that protein phosphorylation on tyrosine residues plays a key role in cellular growth control. This notion has recently received support by the observation that the level of tyrosine phosphorylation of enzymes thought to play an important role in signal transduction (such as phospholipase C) correlates with their increased activity upon growth factor stimulation, thus establishing a functional role for tyrosine phosphorylation (Ullrich, A., et al., Cell 61:203-212 (1990)).
The degree and pattern of phosphorylation of tyrosine residues on cellular proteins are regulated by the opposing activities of protein-tyrosine kinases (PTKases; ATP:protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (PTPases; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). The structural characteristics and evolution of PTKases as well as their role in the regulation of cell growth have been reviewed (Hunter, T., et al., Annu. Rev. Biochem. 54:897-930 (1985); Ullrich, A., et al., supra).
Tyrosine kinases comprise a discrete family of enzymes having common ancestry with, but major differences from, serine/threonine-specific protein kinases (Hanks, S. K. et al., (1988) Science 241, 42-52). The mechanisms leading to changes in activity of tyrosine kinases are best understood for receptor-type tyrosine kinases which have a transmembrane topology (Ullrich, A. et al., supra). With such kinases, the binding of specific ligands to the extracellular domain of these enzymes is thought to induce their oligomerization leading to an increase in tyrosine kinase activity and activation of the signal transduction pathways (Ullrich, A. et al., supra). The importance of this activity is supported by the knowledge that dysregulation of kinase activity through mutation or over-expression is a mechanism for oncogenic transformation (Hunter, T et al., supra; Ullrich, A. et al., 1990, supra).
The protein phosphatases are composed of at least two separate and distinct families (Hunter, T. Cell, 58:1013-1016 (1989)), the protein serine/threonine phosphatases and the protein tyrosine phosphatases. This is in contrast to protein kinases, which show clear sequence similarity between serine/threonine-specific and tyrosine-specific enzymes.
There appear to be two varieties of PTPase molecules. The first group is comprised of small, soluble enzymes that contain a single conserved phosphatase catalytic domain, and include (1) placental PTPase 1B (Charbonneau, H. et al., Proc. Natl. Acad. Sci. 86:5252-5256 (1989); Chernoff, J. et al., Proc. Natl. Acad. Sci. USA 87:2735-2789 (1990)), (2) T-cell PTPase (Cool, D. E. et al., Proc. Natl. Acad. Sci. USA 86:5257-5261 (1989)), and (3) rat brain PTPase (Guan, K., et al., Proc. Natl. Acad. Sci. USA, 87:1501-1505 (1990).
The second group is made up of the more complex, receptor-linked PTPases, termed R-PTPases (or RPTPs), which are of high molecular weight and contain two tandemly repeated conserved domains separated by 56-57 amino acids. One example of RPTPs are the leukocyte common antigens (LCA) (Ralph, S. J., EMBO J., 6:1251-1257 (1987); Charbonneau, H., et al., Proc. Natl. Acad. Sci. USA, 85:7182-7186 (1988)). LCA, also known as CD45, T200 and Ly-5 (reviewed in Thomas, M. L., Ann. Rev. Immunol. 7:339-369 (1989)) comprises a group of membrane glycoproteins expressed exclusively in hemopoietic (except late erythroid) cells, derived from a common gene by alternative splicing events involving the amino terminus of the proteins. Whereas the precise function of CD45 is unknown, many studies have implicated these antigens in a number of processes, including the activity of cytotoxic T lymphocytes and natural killer cells, IL-2 receptor expression, B-cell differentiation, and T lymphocyte proliferation (Pingel, J. T. et al., Cell 58:1055-1065 (1989)).
Other examples of RPTPs are the LCA-related protein, LAR (Streuli, M., et al., J. Exp. Med., 168:1523-1530 (1988)), and the LAR-related Drosophila proteins DLAR and DPTP (Streuli, M., et al., Proc. Natl. Acad. Sci. USA, 86:8698-8702 (1989)). Jirik et al. screened a cDNA library derived from the human hepatoblastoma cell line, HepG2, with a probe encoding the two PTPase domains of LCA (FASEB J. 4:A2082 (1990), abstr. 2253) and discovered a cDNA clone encoding a new RPTP, named He-PTP. The HePTP gene appeared to be expressed in a variety of human and murine cell lines and tissues.
While we are beginning to understand more about the structure and diversity of the PTPases, much remains to be learned about their cellular functions. It has been suggested (Tonks, N. K., et al., Biochemistry, 27:8695-8701 (1988)) that the small, soluble PTPase enzymes may have a xe2x80x9chousekeepingxe2x80x9d function. On the other hand, the RPTPs would be expected to be more restricted in their activities because of their location in the cell membrane and their potential regulation by extracellular ligands. Regarding the role of LCA (CD45) in T cells, it was found that T cell clones deficient in the expression of LCA failed to proliferate when stimulated by a specific antigen or by cross-linking of CD3 (Pingel, J. T., et al., supra). PTPase cross-linking inhibits T cell receptor CD3-mediated activation in human T cells (Kiener, P. A. et al., J. Immunol. 143:23-28 (1989)). The PTPase activity of LCA plays a role in the activation of pp56lck, a lymphocyte-specific PTKase (Mustelin, T., et al., Proc. Natl. Acad. Sci. USA, 86:6302-6306 (1989); Ostergaard, H. L., et al., Proc. Natl. Acad. Sci. USA, 86:8959-8963 (1989)). These authors hypothesized that the phosphatase activity of LCA activates pp56lck by dephosphorylation of a C-terminal tyrosine residue, which may, in turn, be related to T-cell activation.
Using site-directed mutagenesis to determine which of four conserved cysteines in LCA (two per phosphatase domain) was required for enzyme activity toward artificial substrates, Streuli et al. (1989, supra) found that only one cysteine residue (residue 177 of LCA phosphatase domain-1) of LCA was essential for activity, indicating that, most likely, only the first phosphatase domain has enzymatic activity. However, the possibility that the second domain can dephosphorylate a different substrate was not excluded. More recently, Streuli et. al. (EMBO J., 9:2399-2407 (1990)) determined that the second conserved domain of LCA (and of LAR) lacked detectable phosphatase activity but sequences within the domain could influence substrate specificity.
In order to better understand and to be able to control phosphotyrosine metabolism, one must comprehend not only the role of kinase activity, but also the action of phosphatase enzymes as well. Elevation of cellular phosphotyrosine may occur through mechanisms not involving the activation of a tyrosine kinase itself. For instance, expression of the v-crk oncogene, though not a tyrosine kinase itself, induces the phosphorylation of tyrosine residues through a poorly understood mechanism (Mayer, B. J. et al. (1988) Nature 332, 272-275). Potentially, such an outcome could result from either mutation of the substrate or through a general decrease in cellular phosphatase activity, especially in view of the normally high turnover rate of cellular tyrosine-phosphate (Sefton, B. M. et al. (1980) Cell 20, 807-816). The latter possibility is suggested by the demonstration that tyrosine phosphatase inhibitors can xe2x80x9creversibly transformxe2x80x9d cells (Klarlund, J. K. Cell 41: 707-717 (1985)). PTPases could therefore be viewed as potential recessive oncogenes.
It is becoming clear that dephosphorylation of tyrosine can by itself function as an important regulatory mechanism. Dephosphorylation of a C-terminal tyrosine residue stimulates tyrosine kinase activity in the src-family of tyrosine kinases (Hunter, T. (1987) Cell 49, 1-4). Tyrosine dephosphorylation has been suggested to be an obligatory step in the mitotic activation of the MPF (maturation promoting factor) kinase (Morla, A. O. et al. (1989) Cell 58, 193-203). Lastly, mutant analysis of primitive eukaryotes has established crucial roles for serine phosphatase in cellular physiology (Cyert, M. S. et al. (1989) Cell 57, 891-893). These observations point out the need in the art for increasing our understanding of the mechanisms that regulate tyrosine phosphatase activity.
It is clear in the art that further analysis of structure-function relationships among these membrane receptors are needed to gain important understanding of the mechanisms of cell growth, differentiation, and oncogenesis.
The inventors have conceived of a role for RPTPs in cellular control mechanisms, both as potential anti-oncogenes, and as effectors in a newly discovered mechanism of transmembrane signalling. They therefore undertook a search for an RPTP potentially involved in such processes, and describe herein the identification of a novel, widely expressed member of the RPTP family, which has a transmembrane topology. Importantly, its extracellular domain is unrelated to any other RPTP heretofore described. The novel RPTPs, in a manner analogous to receptor tyrosine kinases, are subject to direct regulation by a variety of different extracellular ligands.
The present invention thus provides a human receptor-type protein tyrosine phosphatase (RPTP) protein or glycoprotein molecule other than leucocyte common antigen (LCA or CD45) and leucocyte common antigen-related protein (LAR), a functional derivative of the human RPTP or a homolog of the human RPTP in another mammalian species. When the molecule is of natural origin, it is substantially free of other proteins or glycoproteins with which it is natively associated. This naturally-occurring molecule is normally present in mammalian liver, kidney and brain. Alternatively, the RPTP molecule may not be of natural origin, such as one prepared by chemical or recombinant means.
The substantially pure RPTP protein or glycoprotein of the invention may be produced by biochemical purification of the glycoprotein of natural origin; alternatively, the RPTP may be produced by recombinant means in prokaryotic or eukaryotic hosts.
In particular, the invention is directed to the molecule RPTPxcex1, preferably human RPTPxcex1 having the amino acid sequence (SEQ ID NO:1) shown in FIGS. 4 and 8, or a functional derivative thereof. In another embodiment, the invention is directed to human RPTPxcex2. In yet another embodiment, the invention is directed to human RPTPxcex3.
The invention is further directed to a nucleic acid molecule consisting essentially of a nucleotide sequence encoding RPTPxcex1 of mouse or human origin, or RPTPxcex2 or RPTPxcex3, both of human origin, or a functional derivative thereof. The nucleic acid molecule may be in the form of cDNA or genomic DNA. Preferably, the nucleic acid molecule has the nucleotide sequence of human RPTPxcex1-encoding DNA, SEQ ID NO:2, also shown in FIG. 8. The invention is further directed to the nucleic acid molecule in the form of an expression vehicle, as well as prokaryotic and eukaryotic hosts transformed with the nucleic acid molecule.
Also included in the present invention is a process for preparing an RPTP protein or glycoprotein of this invention, or a functional derivative thereof, comprising:
(a) culturing a host capable of expressing the protein, glycoprotein or functioanl derivative under culturing conditions;
(b) expressing the protein, glycprotein or functional derivative; and
(c) recovering the protein, glycoprotein or functional derivative from the culture.
The invention is directed to an antibody, polyclonal, monoclonal, or chimeric, specific for the RPTPxcex1 protein or glycoprotein.
The invention is also directed to a method for detecting the presence of nucleic acid encoding a normal or mutant RPTP in a subject comprising:
(a) contacting a cell or an extract thereof from the subject with an oligonucleotide probe encoding at least a portion of the normal or mutant RPTP under hybridizing conditions; and
(b) measuring the hybridization of the probe to the nucleic acid of the cell, thereby detecting the presence of the nucleic acid.
The DNA can be selectively amplified, using the polymerase chain reaction, prior to assay.
The invention is further directed to a method for detecting the presence, or measuring the quantity of an RPTP in cell or in a subject comprising:
(a) contacting said cell or an extract thereof with an antibody specific for an epitope of the RPTP; and
(b) detecting the binding of the antibody to the cell or extract thereof, or measuring the quantity of antibody bound, thereby detecting the presence or measuring the quantity of the RPTP.
The present invention is also directed to methods for identifying and isolating a compound capable of binding to an RPTP from a chemical or biological preparation comprising:
(a) attaching the RPTP or the ligand-binding portion thereof to a solid phase matrix;
(b) contacting the chemical or biological preparation with the solid phase matrix allowing the compound to bind, and washing away any unbound material;
(c) detecting the presence of the compound bound to the solid phase; and, for purposes of isolation,
(d) eluting the bound compound, thereby isolating the compound.
Finally, the invention includes a method for identifying a compound capable of stimulating or inhibiting the enzymatic activity of a RPTP, comprising:
(a) contacting the compound with the RPTP in pure form, in a membrane preparation, or in a whole live or fixed cell;
(b) incubating the mixture in step (a) for a sufficient interval;
(c) measuring the enzymatic activity of the RPTP;
(d) comparing the enzymatic activity to that of the RPTP incubated without the compound, thereby determining whether the compound stimulates or inhibits the activity.
In all the above methods, the RPTP is preferably RPTPxcex1, most preferably, human RPTPxcex1.